Functional repair after axonal damage requires disparate responses in the proximal and distal parts of the damaged axon. The proximal axon must initiate new axonal growth, however in many cases this fails to occur, particularly in the adult mammalian CNS. In addition, the distal axonal stump must be cleared out of the way. This occurs via a cell-autonomously initiated axonal fragmentation process termed `Wallerian degeneration'. While this degeneration plays a beneficial role in clearing irreparably damaged axons, the loss of axons is generally a deleterious feature of neuropathies and neurodegenerative diseases. The long term goals of this project are to understand the cellular mechanisms that detect axonal damage and facilitate the dichotomous outcomes of degeneration verses repair. Previous work in the lab, using a Drosophila model, has delineated a conserved molecular pathway that regulates both the regeneration and degeneration of damaged axons. The current grant focuses upon two important components of this pathway: (1) Wnd/DLK, a conserved axonal kinase whose can mediate either regenerative or degenerative responses to axonal injury, hence has been coined a `dual-edged sword', and (2) the NAD+ biosynthetic enzyme, Nmnat [2, 3], whose rapid turnover in distal axons appears to play an important role in initiating degeneration, however the cellular mechanism for Nmnat's protective function(s) is not known. Aim 1 tests a hypothesis, raised by preliminary data, that both Wnd and its DLK homologue in mammals are regulated directly by PKA downstream of cAMP signaling. Because elevated cAMP signaling correlates with successful regeneration in the PNS, this mechanism may yield insight into how pro-regenerative outcomes of Wnd/DLK's activation can be specifically biased. Aim 2 tests a new hypothesis for the protective mechanism in axons, specifically that Nmnat attenuates intracellular Ca2+ influx from ER stores that are triggered by injury, and further links calcium homeostasis to ATP rundown and mitochondrial trafficking in damaged axons. The approaches take advantage of genetic and live imaging techniques in Drosophila larvae which allow for subcellular events (including changes in intracellular Ca2+, ATP, and mitochondrial trafficking) to be manipulated and tracked within injured axons and synapses in live animals. In addition, the project initiates complementary studies in mouse DRG cultures to study axonal regeneration in the context of individual cellular events regulated by DLK. This work is expected to reveal important cellular mechanisms that influence both regenerative and degenerative responses to axonal damage.